Yolk-derived hypoglycemic peptide as well as preparation method and application thereof

Abstract

The invention discloses a yolk-derived hypoglycemic peptide as well as a preparation method and application thereof, and belongs to the technical field of polypeptides. The amino acid sequence of the yolk source blood sugar lowering peptide is YVIQEDR. The novel peptide YVIQEDR capable of reducing blood sugar is identified and obtained from the degreased egg yolk powder hydrolysate by utilizing a molecular docking technology, and compared with the degreased egg yolk powder hydrolysate, the peptide YVIQEDR is proved to have higher blood sugar reducing activity. Besides, the YVIQEDR also has the advantages of no toxicity, high safety, strong activity and the like, can be used as an ideal effective factor for reducing blood sugar, can be widely applied to the fields of food, health care products, medicines and the like, and has a huge application value.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to an egg yolk-derived blood sugar-lowering peptide and a preparation method and application thereof. Background Art

[0002] Diabetes is a common chronic metabolic disease. Long-term hyperglycemia can lead to numerous complications and severely harm human health. Currently, diabetes treatment relies primarily on medication and insulin injections, but these treatments can cause a range of side effects with long-term use. Therefore, developing safe and effective alternative therapies for lowering blood sugar has become a research hotspot.

[0003] Carbohydrates are the primary energy source in the human diet, and their digestion has a significant impact on blood glucose regulation. The digestion of carbohydrates into free glucose primarily relies on the combined action of α-amylase and α-glucosidase. α-amylase initially hydrolyzes carbohydrates into disaccharides and oligosaccharides. Upon reaching the brush border of the small intestine, α-glucosidase efficiently catalyzes the breakdown of α-1,4- and α-1,6-glycosidic bonds into absorbable glucose. Due to its intestinal targeting, α-glucosidase is considered an ideal target for the development of safe and effective hypoglycemic drugs. Inhibiting α-glucosidase activity can delay the conversion of polysaccharides and disaccharides into absorbable glucose, thereby reducing postprandial blood glucose peaks and helping to control blood glucose fluctuations. This has significant therapeutic benefits for patients with early-stage diabetes and impaired glucose tolerance, and is also suitable for some patients with insulin resistance. Furthermore, α-glucosidase inhibitors have the advantage of not stimulating insulin secretion and therefore not causing hypoglycemia, which is crucial for avoiding blood glucose fluctuations and maintaining blood glucose stability throughout the day. Acarbose, one of the first α-glucosidase inhibitors introduced to the market in 1990, remains the most prescribed drug to date. However, common adverse reactions, such as flatulence, abdominal pain, and diarrhea, limit the tolerability of long-term use. Therefore, the discovery of safe and effective α-glucosidase inhibitory peptides from natural food sources has attracted widespread attention. These bioactive peptides are derived from foods that are safe to consume over a long period of time, generally have good biocompatibility, and are less likely to cause serious side effects.

[0004] In recent years, the research on functional foods and natural active peptides has gradually become a hot topic in the field of diabetes prevention and treatment. Food-derived functional peptides are active peptides released through enzymatic hydrolysis from natural food sources such as dairy products, soybeans, fish, eggs, etc., and have a wide range of biological activities. Egg yolk, as a nutrient-dense natural raw material, is rich in high-quality protein, which can release functional peptides after proper enzymatic hydrolysis. However, there are relatively few studies on specific peptides with hypoglycemic activity in egg yolk protein hydrolysates, and there is still a lack of clear structure-function relationship and target mechanism research. Therefore, the development of a functional peptide derived from egg yolk with significant hypoglycemic activity has become an urgent need for diabetic nutritional intervention and functional food development. Summary of the Invention

[0005] The present invention aims to provide an egg yolk-derived hypoglycemic peptide, a preparation method, and an application thereof, in order to solve the problems existing in the above-mentioned prior art. The present invention utilizes molecular docking-based directional screening technology to obtain a highly effective hypoglycemic peptide, YVIQEDR, from defatted egg yolk powder hydrolysate. The peptide can be used in industries such as food, health products, medicine, and animal feed, and has broad application prospects.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an egg yolk-derived hypoglycemic peptide, wherein the amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR.

[0008] The present invention also provides a method for preparing the egg yolk-derived hypoglycemic peptide, comprising the following steps:

[0009] The defatted egg yolk powder is added with water and enzymatically hydrolyzed with a single protease, and after the enzymatic hydrolysis, the enzyme is inactivated, cooled, and centrifuged to collect the supernatant to obtain the defatted egg yolk powder hydrolyzate;

[0010] The defatted egg yolk powder hydrolyzate is separated into peptide segments by HPLC liquid chromatography;

[0011] The peptide segment was docked with α-glucosidase through molecular simulation, and the egg yolk-derived hypoglycemic peptide YVIQEDR was screened and identified.

[0012] Optionally, the single protease comprises papain, alkaline protease, trypsin or neutral protease.

[0013] Preferably, the single protease is trypsin.

[0014] Optionally, the amount of enzyme added for the enzymatic hydrolysis is 2000 U / g, and the enzymatic hydrolysis time is 2 hours.

[0015] Optionally, the centrifugation is performed at 4°C and 8000 rpm for 15 min.

[0016] Optionally, the mobile phase A of the HPLC liquid chromatography is 0.1% formic acid, and the mobile phase B is a mixture of 0.1% formic acid and 99.9% acetonitrile.

[0017] The present invention also provides the use of the egg yolk-derived hypoglycemic peptide in the preparation of a drug for lowering blood sugar.

[0018] The present invention also provides the use of the egg yolk-derived hypoglycemic peptide in the preparation of health products that help maintain healthy blood sugar levels.

[0019] The present invention also provides a blood sugar lowering drug comprising the egg yolk-derived blood sugar lowering peptide.

[0020] The present invention also provides a health product that helps maintain a healthy blood sugar level, comprising the egg yolk-derived hypoglycemic peptide.

[0021] The present invention discloses the following technical effects:

[0022] Using molecular docking technology, this study identified a novel hypoglycemic peptide, YVIQEDR, from defatted egg yolk hydrolysate. This peptide has been shown to exhibit stronger hypoglycemic activity than defatted egg yolk hydrolysate. Furthermore, YVIQEDR offers advantages such as non-toxicity, high safety, and strong activity, making it an ideal hypoglycemic agent for a wide range of applications in food, healthcare products, and pharmaceuticals, demonstrating its enormous potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 is the secondary mass spectrum of peptide YVIQEDR;

[0025] Figure 2 Schematic diagram of the docking of peptide YVIQEDR and α-glucosidase molecule;

[0026] Figure 3 for Figure 2 The interaction between the black-boxed peptide YVIQEDR and α-glucosidase; the green dashed lines represent hydrogen bonds, and the arcs represent residues involved in hydrophobic interactions;

[0027] Figure 4 To evaluate the in vitro hypoglycemic activity of the peptide YVIQEDR. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1 Preparation and screening of polypeptides

[0034] 1. Enzymatic hydrolysis

[0035] With defatted egg yolk powder (purchased from Baiyunshan Pharmaceutical Factory of Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd.) as raw material, double distilled water was added according to the volume ratio of powder: water = 1:10, and single protease (including papain, alkaline protease, trypsin and neutral protease, the enzyme used in this embodiment is trypsin) was used for enzymolysis, the enzyme amount was 2000U / g, the pH of the solution was adjusted to 8 (the suitable pH of the enzyme), and after mixing, it was placed in a constant temperature shaking water bath at a temperature of 37°C for enzymolysis. The enzymolysis time was 2 hours, and the pH was adjusted to 8 every 1 hour during the enzymolysis. After the enzymolysis was completed, the enzymolysis solution was inactivated in a boiling water bath for 10 minutes, then cooled to room temperature and centrifuged at 8000rpm for 15 minutes at 4°C, and the supernatant (i.e., the defatted egg yolk powder hydrolyzate) was collected and stored for later use.

[0036] 2. Peptide identification

[0037] The amino acid sequences of peptides in defatted egg yolk powder hydrolysate were identified by LC-MS / MS.

[0038] The hydrolyzate was separated using a nanoElute HPLC system with a nanoflow rate. The column information was a Thermo Scientific EASY column, 25 cm, ID 75 μm, 1.9 μm, C18; mobile phase A: 0.1% formic acid, mobile phase B: a mixture of 0.1% formic acid and 99.9% acetonitrile (ACN); flow rate: 300 nL / min; the liquid phase gradient was as follows: 0 min - 18 min, linear gradient of phase B from 5% to 35%; 18 min - 20 min, linear gradient of phase B from 35% to 80%; 20 min - 30 min, phase B maintained at 80%.

[0039] After chromatographic separation, the samples were analyzed by mass spectrometry using a timsTOF Pro mass spectrometer. The ion source voltage was set to 1.5 kV, and TOF was used for both MS and MSMS detection and analysis. The mass spectrometer scan range was set from 100 m / z to 1700 m / z. Data acquisition was performed in parallel accumulation serial fragmentation (PASEF) mode. After the primary mass spectrometer was acquired, eight PASEF acquisitions were performed in the parent ion mode with a cycle window of 0.95 s and a secondary spectrum with a charge number range of 0-5. The dynamic exclusion time of the tandem mass spectrometer scan was set to 24 s to avoid duplication of parent ions.

[0040] 3. Peptide screening: Select peptides with a molecular weight of less than 1 kDa, a length of 2-10 amino acid residues, non-toxicity and good water solubility from the numerous matched peptide sequences.

[0041] 4. Molecular docking

[0042] Molecular docking was performed using AutoDock software. The specific operations are as follows:

[0043] First, the receptor α-glucosidase was prepared. The α-glucosidase molecule was derived from the RCSB database (3WY1). Water molecules and ligand small molecules were deleted, and hydrogen and charge were added. Then, the ligand was prepared and the three-dimensional structure of the screened peptide was drawn using the peptide structure online generation website (https: / / cloud.yinfotek.com / console / ). Molecular docking was performed using Auto Dock Vina, and the center coordinates of the active pocket were set to x = -6.665, y = -12.514, z = 21.514. The docking box size was set to 80 × 80 × 80. The binding energy between the peptide and α-glucosidase was obtained. Among them, the peptide segment YVIQEDR (Tyr-Val-Ile-Gln-Glu-Asp-Arg) had the best molecular docking effect, with a binding energy of -8.4 kcal / mol. The secondary mass spectrum of YVIQEDR is shown in Figure 1 The schematic diagram of its docking with α-glucosidase molecule is shown in Figure 2 , see interaction Figure 3 ,Depend on Figure 2-3 It can be seen that the peptide YVIQEDR interacts with α-glucosidase through hydrogen bonds, hydrophobic interactions and salt bridges.

[0044] Example 2 Determination of hypoglycemic activity of active polypeptides

[0045] The peptide YVIQEDR obtained by screening was synthesized in vitro at a concentration of 2 mg / mL. The hypoglycemic activity of the peptide was evaluated using the defatted egg yolk powder hydrolyzate prepared in Example 1 as a control.

[0046] α-glucosidase inhibition rate determination method: The substrate p-nitrophenylpyranoside and α-glucosidase are dissolved in phosphate buffer (0.1M, pH 6.8). The sample is mixed with 0.5U / mL α-glucosidase and incubated at 37°C for 15 minutes. 2.5mM substrate solution is added to the mixture, and the mixture is incubated at 37°C for another 20 minutes. Finally, 1M Na2CO3 is added to terminate the reaction. The control group uses phosphate buffer instead of sample solution, and the blank group uses phosphate buffer instead of enzyme solution. The absorbance is measured at 405nm and calculated according to the following formula.

[0047]

[0048] Where: C is the absorbance value without sample, A is the absorbance value with sample added, and B is the blank control of A.

[0049] The results of hypoglycemic activity are shown in Figure 4The results showed that the inhibition rate of 2 mg / mL YVIQEDR on α-glucosidase reached 64.36%, which was significantly higher than that of defatted egg yolk powder hydrolysate (37.56%, P<0.05), verifying the hypoglycemic effect of YVIQEDR.

[0050] Based on the above results, it can be seen that YVIQEDR can interact with α-glucosidase through hydrogen bonds, hydrophobic interactions and salt bridges, thereby inhibiting the activity of α-glucosidase and exerting a hypoglycemic effect.

[0051] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An egg yolk-derived hypoglycemic peptide, characterized in that: The amino acid sequence of the egg yolk-derived hypoglycemic peptide is YVIQEDR.

2. The method for preparing the egg yolk-derived hypoglycemic peptide according to claim 1, wherein: The following steps are involved: The defatted egg yolk powder is added with water and enzymatically hydrolyzed with a single protease, and after the enzymatic hydrolysis, the enzyme is inactivated, cooled, and centrifuged to collect the supernatant to obtain the defatted egg yolk powder hydrolyzate; The defatted egg yolk powder hydrolyzate is separated into peptide segments by HPLC liquid chromatography; The peptide segment was docked with α-glucosidase through molecular simulation, and the egg yolk-derived hypoglycemic peptide YVIQEDR was screened and identified.

3. The preparation method according to claim 2, wherein The single protease includes papain, alkaline protease, trypsin or neutral protease.

4. The preparation method according to claim 2, wherein The enzyme dosage for the enzymatic hydrolysis was 2000 U / g, and the enzymatic hydrolysis time was 2 hours.

5. The preparation method according to claim 2, wherein The centrifugation was carried out at 4°C and 8000 rpm for 15 min.

6. The preparation method according to claim 2, wherein The mobile phase A of the HPLC liquid chromatography is 0.1% formic acid, and the mobile phase B is a mixture of 0.1% formic acid and 99.9% acetonitrile.

7. Use of the egg yolk-derived hypoglycemic peptide according to claim 1 in the preparation of a drug for lowering blood sugar.

8. Use of the egg yolk-derived hypoglycemic peptide according to claim 1 in the preparation of a health product that helps maintain healthy blood sugar levels.

9. A blood sugar lowering drug, characterized in that: The invention comprises the egg yolk-derived hypoglycemic peptide according to claim 1.

10. A health product that helps maintain healthy blood sugar levels, characterized in that: The invention comprises the egg yolk-derived hypoglycemic peptide according to claim 1.

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